Prostanoids can be assayed by the highly sensitive and reproducible gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) technique. This paper describes a simplified method applied to the quantification of prostaglandins (PG) in the supernatants of limited amounts of cells, cultured in multiwell plates (30,000 cells/well). The culture medium was extracted on C2 columns and the derivatization was performed in three steps. Prostanoids were then analyzed by GC-NICI-MS within 10 min. The storage conditions at each step were tested in order to achieve maximal yields. This procedure allowed the determination and quantification of PG produced by cultured human synovial cells and chondrocytes at the basal level as well as after interleukin-1 stimulation. PGE2, PGF2 alpha, and 6-keto-PGF1 alpha, the stable form of PGI2, were the main PG detected. Derivatization and GC-MS analyses of 18 samples could easily be performed in 1 day. The simplified GC-NICI-MS method can thus be routinely applied for assaying PG in any cell line cultured in multiwells.